QuantiTect Whole Transcriptome Kit

对珍贵的RNA样本进行无限次real-time PCR分析

  • 高cDNA产量用于无限次分析和长期保存
  • 均一扩增所有cDNA和所有转录本区段
  • 经优化的试剂,快速简单的操作流程

QuantiTect Whole Transcriptome Kit对有限的RNA样品进行预扩增和逆转录,获得高产量cDNA,用于无限次real-time PCR基因表达分析。该试剂盒包含全转录扩增所需的全部酶和缓冲液。对Phi29聚合酶创新的修饰确保从低至1 ng RNA中制备多至40 μg cDNA。该聚合酶独特的加工过程可制备高度统一的cDNA,确保通过real-time PCR进行可靠的基因表达分析。

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QuantiTect Whole Transcriptome Kit (25)
For 25 x 50 µl reactions: 25 µl T-Script Enzyme, 400 µl T-Script Buffer, 25 µl Ligation Enzyme 1, 25 µl Ligation Enzyme 2, 200 µl Ligation Reagent, 600 µl Ligation Buffer, 25 µl REPLI-g Midi DNA Polymerase, 730 µl REPLI-g Midi Reaction Buffer
207043
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QuantiTect Whole Transcriptome Kit (100)
For 100 x 50 µl reactions: 100 µl T-Script Enzyme, 400 µl T-Script Buffer, 100 µl Ligation Enzyme 1, 100 µl Ligation Enzyme 2, 200 µl Ligation Reagent, 600 µl Ligation Buffer, 100 µl REPLI-g Midi DNA Polymerase, 2 x 1.45 ml REPLI-g Midi Reaction Buffer
207045
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The QuantiTect Whole Transcriptome Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Reproducible cDNA yields.|Equal amplification of 5' and 3' regions.|Procedure.|Reliable real-time PCR analysis.|Whole transcriptome amplification.|Preservation of transcript profile.|
Total RNA (10 ng) from different blood samples was amplified for 2 or 8 hours using the QuantiTect Whole Transcriptome Kit. cDNA yields were determined using PicoGreen reagent.|Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit. This was followed by real-time PCR using 10 ng cDNA, primers specific for β-actin, and the QuantiTect SYBR® Green PCR Kit. Amplicons corresponding to the 5' and 3' regions of the β-actin transcript were detected with similar CT values, indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions.|cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.|Replicate RNA samples (10 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR of the indicated targets was then performed using 10 ng cDNA and the QuantiFast Probe PCR Kit on the Mx3005P system. The overlapping curves indicate highly reproducible whole transcriptome amplification. ACTB: β-actin; HPRT1: Hypoxanthine phosphoribosyltransferase 1.|cDNA is first synthesized from template RNA and then ligated (not shown). REPLI-g DNA polymerase moves along the cDNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, allowing high yields of cDNA to be generated.|Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit for 2 hours (WTA 2 h) or 8 hours (WTA 8 h), followed by real-time PCR of 5 targets using 10 ng cDNA. As a control, RNA was reverse transcribed without amplification using the Sensiscript RT Kit (Non-WTA), and 1/5 of the RT reaction was used in real-time PCR. Real-time PCR was carried out using the QuantiTect Probe PCR Kit. [A] Relative gene expression levels of 5 transcripts (normalization to HPRT1) are similar in the WTA 2 h, WTA 8 h, and Non-WTA samples, as shown by the overlapping points. [B] A plot of ΔCT values (CT value for each target minus CT value for internal reference β-actin) for WTA 8 h samples (Y-axis) against those for Non-WTA samples (X-axis) is highly linear. The data in [A] and [B] demonstrate that the QuantiTect Whole Transcriptome Kit preserves the transcript profile. TP53: Tumor protein p53; NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; HPRT1: hypoxanthine phosphoribosyltransferase 1; TBP: TATA box binding protein; TNFRSF6B: tumor necrosis factor receptor superfamily, member 6b, decoy.|
Performance

QuantiTect Whole Transcriptome Kit确保所有转录子高度一致的扩增,这对于基因表达分析是必需的。所有mRNA转录子以相同的形式同时在5'和3'末端扩增(参见"Equal amplification of 5' and 3' regions")。图"Preservation of transcript profile", 显示高度稳定的转录子,该图将分别应用QuantiTect Whole Transcriptome Kit全基因组扩增的cDNA和用逆转录酶制备的未扩增cDNA进行了对比。

可获得多至40 μg的可重复、高产量的cDNA(参见"Reproducible cDNA yields")。能够进行无限次real-time PCR分析,并具有高度重复性的CT值(参见"Reliable real-time PCR analysis")。此外,由于cDNA比RNA更稳定,可以存档用于以后的研究工作。

高产量的cDNA可用于real-time PCR分析
起始材料 扩增时间 扩增产物的常规产量 可进行的real-time PCR次数*
10 ng RNA 2小时 至多10 µg cDNA 1000
10 ng RNA 8小时 至多40 µg cDNA 4000
* 从一次QuantiTect whole transcriptome amplification (WTA)扩增反应得到的cDNA可进行的real-time PCR次数。不使用WTA,10 ng RNA只能进行一次可靠的real-time RT-PCR分析。
Principle

获得的生物样本量太少可能限制基因表达谱分析。使用QuantiTect Whole Transcriptome Kit可对少量珍贵的样品进行无限次real-time PCR分析。QuantiTect Whole Transcriptome Kit经优化,用于1 ng RNA或相当于约50个细胞RNA的全转录扩增,甚至可以用更少量的RNA,这些取决于RNA的品质和转录子的丰度。

QuantiTect Whole Transcriptome Kit含有逆转录酶、DNA聚合酶和优化的缓冲液及试剂,可对一个RNA样本的所有转录本进行扩增。此试剂盒整合了经质量验证的REPLI-g技术,通过非偏向序列扩增来合成cDNA。REPLI-g扩增采用Multiple Displacement Amplification(MDA)技术,应用独特加工的DNA聚合酶实现等温扩增(参见"Whole transcriptome amplification")。该技术确保包括5’末端在内的所有转录区的同等扩增,从而获得可用于real-time PCR的足够多的cDNA。

Procedure
从总RNA开始经过3个步骤可获得大量代表了整个转录子的cDNA(参见"Procedure")。首先,应用优化的含有T-Script Enzyme的逆转录混合物(含有随机引物和oligo-dT引物)将RNA转录为cDNA。用高效的连接混合物连接,然后用基于经验证的REPLI-g技术的扩增混合物扩增cDNA。
Applications

QuantiTect Whole Transcriptome Kit制备的cDNA可用于QuantiFast或Rotor-Gene Kits进行的real-time PCR分析,且可以储存用于以后的分析,但不适用于微阵列分析。QIAGEN提供REPLI-g Kits and Service,用于小量或珍贵样品的全基因组扩增。扩增高度一致,具有最小的序列偏好,可制备适合于基因分型和Comparative Genome Hybridization等应用的DNA。

Feature
Specifications
Amplification Whole total RNA
Applications Real-time PCR, gene expression analysis
Maximum input volume 5 µl total RNA
Minimal pipetting volume needed 1 µl
Reaction time 5 hours or 11 hours
Reaction volume 50 µl
Samples per run; throughput Mid
Starting amount of total RNA >10 ng purified total RNA
Starting material Purified total RNA
Technology Reverse transcription followed by ligation and multiple displacement amplification (MDA)
Yield 10 µg or 40 µg cDNA

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Kit Handbooks
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For preparation of cDNA from total RNA by whole transcriptome amplification
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